Abstract

For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of "the dose makes the poison," because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.

Characteristics and activities of natural hormones. A, This schematic depicts a typical relationship of three phases of circulating hormones: free (the active form of the hormone), bioavailable (bound weakly to proteins such as albumin), and inactive (bound with high affinity to proteins such as SHBG). These three phases act as a buffering system, allowing hormone to be accessible in the blood, but preventing large doses of physiologically active hormone from circulating. With EDCs, there may be little or no portion maintained in the inactive phase. Thus, the entirety or majority of a circulating EDC can be physiologically active; the natural buffering system is not present, and even a low concentration of an EDC can disrupt the natural balance of endogenous hormones in circulation. B, Schematic example of the relationship between receptor occupancy and hormone concentration. In this theoretical example, at low concentrations, an increase in hormone concentration of x (from 0 to 1x) causes an increase in receptor occupancy of approximately 50% (from 0 to 50%, see yellow box.) Yet the same increase in hormone concentration at higher doses (from 4x to 5x) causes an increase in receptor occupancy of only approximately 4% (from 78 to 82%, see red box).

Intrauterine position produces offspring with variable circulating hormone levels. Fetuses are fixed in position in the bicornate rodent uterus, thus delivery via cesarean section has allowed for study of the influence of intrauterine position on behaviors, physiology, and organ morphology. Illustrated here are the differences in estradiol (E2) and testosterone (T) concentrations measured in male and female fetuses positioned between two male neighbors (2M), two female neighbors (2F), or neighbors of each sex (1MF). Direction of blood flow in the uterine artery (dark vessel) and vein (light vessel) is indicated by an arrow ().

Examples of dose-response curves. A, Linear responses, whether there are positive or inverse associations between dose and effect, allow for extrapolations from one dose to another. Therefore, knowing the effects of a high dose permits accurate predictions of the effects at low doses. B, Examples of monotonic, nonlinear responses. In these examples, the slope of the curve never changes sign, but it does change in value. Thus, knowing what happens at very high or very low doses is not helpful to predict the effect of exposures at moderate doses. These types of responses often have a linear component within them, and predictions can be made within the linear range, as with other linear responses. C, Displayed are three different types of NMDRCs including an inverted U-shaped curve, a U-shaped curve, and a multiphasic curve. All of these are considered NMDRCs because the slope of the curve changes sign one or more times. It is clear from these curves that knowing the effect of a dose, or multiple doses, does not allow for assumptions to be made about the effects of other doses. D, A binary response is shown, where one range of doses has no effect, and then a threshold is met, and all higher doses have the same effect.

NOAEL, LOAEL, and calculation of a safe reference dose. A, In traditional toxicology testing, high doses are tested to obtain the maximum tolerated dose (MTD), the LOAEL, and the NOAEL. Several safety factors are then applied to derive the reference dose, i.e. the dose at which exposures are presumed safe. This reference dose is rarely tested directly. Yet when chemicals or hormones produce NMDRCs, adverse effects may be observed at or below the reference dose. Here, the doses that would be tested are shown by a dotted line, and the calculated safe dose is indicated by a thick solid line. The actual response, an inverted U-shaped NMDRC, is shown by a thin solid line. B, Experimental data indicate that EDCs and hormones do not have NOAELs or threshold doses, and therefore no dose can ever be considered safe. This is because an exogenous hormone (or EDC) could have a linear response in the tested range (dotted line), but because endogenous hormones are present (thin solid line), the effects of the exogenous hormone are always observed in the context of a hormone-containing system.

Example of a NMDRC in humans and the sampling populations that could be examined in epidemiology studies. This schematic illustrates a theoretical NMDRC in a human population. If a study were to sample only group A, the conclusion would be that with increasing exposures, risk increases monotonically. Sampling group B would allow researchers to conclude that there is a nonmonotonic relationship between exposure level and risk. If a study included only group C, the conclusion would be that with increasing exposures, there is decreased risk of disease. Group D represents a population that was highly exposed, i.e. due to an industrial accident. This group has the highest risk, and there is a monotonic relationship between exposures and risk, although risk is high for all individuals. In the group D situation, there is generally a background population with which high-dose exposure is compared (dotted line); relative risk for group D would depend on whether that background population resembles group A, B, or C. From this example, it is clear that the population sampled could strongly influence the shape of the dose-response curve produced as well as the conclusions reached by the study.

Dose-response ranges for tamoxifen in breast cancer therapy. This figure demonstrates the NMDRC, also called flare, in tamoxifen treatments. As the circulating dose of tamoxifen increases when treatment starts, patients initially experience flare, i.e. growth of the tumor (), followed by a decrease in tumor size as the circulating levels of tamoxifen rise into the therapeutic range (, ). High doses of tamoxifen are acutely toxic (). Starting from the highest concentrations, where acute toxicity is observed, and going to lower concentrations on the X-axis, the acute toxicity diminishes towards zero growth, i.e. therapeutic stasis (green baseline). This occurs at approximately 1E-05 m, the lowest observed effect level (LOEL) for toxicity. The vertical arrows show the results of applying three or four 10-fold safety factors to the LOEL for the high-dose toxicity of tamoxifen, and would calculate a safe or reference dose for tamoxifen in the region of flare, the least safe region of exposure in actual practice. Above the diagram of dose response ranges is estimated ER occupancy by tamoxifen. This was calculated from the affinity constant of tamoxifen for ERs determined in human breast cancer cells (Ki = 29.1 nM; Ref 678); flare appears to correspond to low receptor occupancy (blue axis), therapeutic range with mid and upper-range receptor occupancy, and acute toxicity well above 99% receptor occupancy. ().

Leptin as an example of a NMDRC. Several studies report NMDRCs in response to leptin treatments. A, NMDRCs are observed in cultured primary adipocytes after leptin exposure. This graph illustrates the relationship between administered leptin dose and glucose uptake in two types of adipocytes, those isolated from omental tissue (green) and others from sc fat (purple) (schematic was made from data in Ref. ). These data are on a log-linear plot. B, Ex vivo rat pancreas was treated with leptin and various doses of glucose, and the insulin response curves were examined. Area under the curve is a measure of the ability of the pancreas to bring glucose levels under control. Different dose-response curves were observed depending on the amount of glucose administered: a U-shaped curve when 8 mmol/liter was included (pink) or a multiphasic curve with 4 mmol/liter (blue) (schematic made from data in Ref. ). These data are on a linear-linear plot. C, U-shaped NMDRCs were also observed when food intake was compared with leptin levels in the blood of rats administered the hormone. This response was similar in males (orange) and females (cyan) (schematic made from data in Ref. ). These data are on a linear-linear plot.